I have proposed in International Patent Application WO84/01031 to measure the concentration of an analyte in a fluid by contacting the fluid with a trace amount of a binding agent such as an antibody specific for the analyte in the sense that it reversibly binds the analyte but not other components of the fluid, determining a quantity representative of the proportional occupancy of binding sites on the binding agent and estimating from that quantity the analyte concentration. In that application I point out that, provided that the amount of binding agent is sufficiently low that its introduction into the fluid causes no significant diminution of the concentration of ambient (unbound) analyte, the fractional occupancy of the binding sites on the binding agent by the analyte is effectively independent of the absolute volume of the fluid and of the absolute amount of binding agent, i,e. independent within the limits of error usually associated with the measurement of fractional occupancy. In such circumstances, and in these circumstances only, the initial concentration [H] of analyte in the fluid is related to the fraction (Ab/Ab.sub.o) of binding sites on the binding agent occupied by the analyte by the equation: EQU Ab/Ab.sub.o =K.sub.ab [H]/1+K.sub.ab [H]
where K.sub.ab (hereinafter referred to as K) is the equilibrium constant for the binding of the analyte to the binding sites and is a constant for a given analyte and binding agent at any one temperature. This constant is generally known as the affinity constant, especially when the binding agent is an antibody, for example a monoclonal antibody.
The concept of using only a trace amount of binding agent is contrary to generally recommended practice in the field of immunoassay and immunometric techniques. For example, in such a well-known work as "Methods in Investigative and Diagnostic Endocrinology", ed. S. A. Berson and R. S. Yalow, 1973 at pages 111-116, it is proposed that in the performance of a competitive immunoassay maximum sensitivity of the assay is achieved if the proportion of the "tracer" analyte that is bound approximates to 50%. In order to achieve such a high degree of binding of the analyte the theory of Berson and Yalow, to this day generally accepted by other workers in the field, requires that the concentration of binding agent (or, strictly speaking, of binding sites, each molecule of binding agent conventionally having one or at most two binding sites) must be greater than or equal to the reciprocal of the equilibrium constant (K) of the binding agent for the analyte, i.e. [ab]&gt;1/K. For a sample of volume V the total amount of binding agent (or binding sites) must therefore be greater than or equal to V/K. A binding agent which is a monoclonal antibody may, for example, have an equilibrium constant (K) which is of the order of 10.sup.11 liters/mole for the specific antigen to which it binds. Thus, under the above generally accepted practice, a binding agent (or site) concentration of the order of 10.sup.-11 mole/liter or more is required for binding agents of such an equilibrium constant and, with fluid sample volumes of the order of 1 milliliter, the use of 10.sup.-14 or more mole of binding agent (or site) is conventionally deemed necessary. Avogadro's number is about 6.times.10.sup.23 so that 10.sup.-14 mole of binding site is equivalent to more than 10.sup.9 molecules of binding agent even assuming that the binding agent possesses two binding sites per molecule. For specific binding agents of the very highest affinity K is less than 10.sup.13 liters/mole so that conventional practice requires more than 10.sup.7 molecules of binding agent, whereas binding agents with lower affinity of the order of 10.sup.8 liters/mole necessitate the use of more than 10.sup.12 molecules under conventional practice. In fact all immunoassay kits marketed commercially at the present time conform to these concepts and use an amount of binding site approximating to or, more frequently, considerably in excess of V/K; indeed in certain types of kit relying on the use of labelled antibodies it is conventional to use as much binding agent as possible, binding proportions of analyte greatly exceeding 50 %.
Because of the binding of substantial proportions, for example 50%, of the analyte in the liquid samples under test in such systems, the fractional occupancy of the binding sites of the binding agent is not independent of the volume of the fluid sample so that for accurate quantitative assays it is necessary to control accurately the volume of the sample, keeping it constant in all tests, whether of the sample of unknown concentration or of the standard samples of known concentration used to generate the dose response curve. Furthermore, such systems also require careful control of the amount of binding agent present in the standard and control incubation tubes. These limitations of present techniques are universally recognised and accepted.
UK Patent Application 2,099,578A discloses a device for immunoassays comprising a porous solid support to which antigens, or less frequently immunoglobulins, are bound at a plurality of spaced apart locations, said device permitting a large number of qualitative or quantitative immunoassays to be performed on the same support, for example to establish an antibody profile of a sample of human blood serum. However, although the individual locations may be in the form of so-called microdots produced by supplying droplets of antigen-containing solutions or suspensions, the number of moles of antigen present at each location is apparently still envisaged as being enough to bind essentially all of the analyte (e.g. antibody) whose concentration is to be measured that is present in the liquid sample under test. This is apparent from the fact that the quantitative method used in that application (page 3, lines 21-28) involves calibration with known amounts of immunoglobulin being applied to the support; but this means that, in the samples being tested, essentially every molecule must be extracted from the sample in order for a true comparison to be made and hence that large amounts of antigen (i.e. the binding agent in this situation) are required in each microdot, greatly in excess of the total amount of analyte (i.e. antibody in this situation) present in the sample.